Response surface methodology
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Microbial transglutaminase (MTG) is an enzyme widely used in the food industry because it creates cross-links between proteins, enhancing the texture and stability of food products. Its unique properties make it a valuable tool for modifying the functional characteristics of proteins, significantly impacting the quality and innovation of food products. In this study, response surface methodology was employed to optimize the fermentation conditions for microbial transglutaminase production by the strain Streptoverticillium cinnamoneum KKP 1658. The effects of nitrogen dose, cultivation time, and initial pH on the activity of the produced transglutaminase were investigated. The significance of the examined factors was determined as follows: cultivation time > nitrogen dose > pH. The interaction between nitrogen dose and cultivation time was found to be crucial, having the second most significant impact on transglutaminase activity. Optimal conditions were identified as 48 h of cultivation with a 2% nitrogen source dose and an initial medium pH of approximately 6.0. Under these conditions, transglutaminase activity ranged from 4.5 to 5.5 U/mL. The results of this study demonstrated that response surface methodology is a promising approach for optimizing microbial transglutaminase production. Future applications of transglutaminase include the development of modern food products with improved texture and nutritional value, as well as its potential use in regenerative medicine for creating biomaterials and tissue scaffolds. This topic is particularly important and timely as it addresses the growing demand for innovative and sustainable solutions in the food and biomedical industries, contributing to an improved quality of life.
PURPOSE: While anal cancer is a very rare oncological diagnosis representing less than 2% of lower gastrointestinal tract cancers, the incidence has doubled in the past 20 years. Radical radiochemotherapy with sequential or simultaneous boost is now the standard treatment modality. Interstitial HDR brachytherapy is one of the boost application options. Implementation of new radiotherapy techniques has resulted in improved therapeutic outcomes; however, it is still associated with acute and especially late toxicity. Gastrointestinal disorders and sexual dysfunction are the most frequent factors affecting the long-term quality of cured patients' lives. METHODS: A total of 96 patients consecutively treated between 2000 and 2022 with external beam radio-/chemotherapy and an interstitial brachytherapy boost for histologically verified nonmetastatic anal squamous cell carcinoma were evaluated. The median follow-up time was 15.4 years (range 13.4-17.3 years). The primary objective of the study was to assess local control (LC) and quality of life (QoL). The Czech versions of internationally validated EORTC questionnaires were used to evaluate life quality-the basic EORTC QOL-C30 v.3 and the specific QOL-ANL 27 questionnaire. RESULTS: Local control was 85.5% at 5 years, 83.4% at 10 years, 83.4% at 15 years, and 83.4% at 20 years, and there was no dependence on clinical stage. The most common forms of acute toxicity were cutaneous and hematological but were gastrointestinal for late toxicities. In the evaluation of quality of life, 80.5% of patients alive at the time participated. In the EORTC quality of life questionnaire C30 v.3, patients rated the functional scale score as 86.2 points (standard deviation [SD] = 12.6) and the symptom score as 15.5 points (SD = 12.5). The global health score achieved 68.4 points (SD = 23.6). The most common symptoms were fatigue with 25.6 points (SD = 20.2) and diarrhea with 19.0 points (SD = 27.8). In the QOL-ANL 27 questionnaire, symptom scales assessing bowel symptoms were scored 27.5 points (SD = 19) in non-stoma patients and 11.9 points (SD = 17.2) in stoma patients. In the single-item symptom scales, the highest scores were rated for frequency of urination with 26.4 points (SD = 30.8), need to be close to a toilet with 22.4 points (SD = 27.3), and self-cleaning more often with 25.3 points (SD = 31.8). In the functional scales assessing sex life and interest, men and women reported scores of 45.2 (SD = 23) and 45.5 points (SD = 19), respectively. CONCLUSION: Boost with interstitial HDR brachytherapy is an established safe method of anal cancer treatment, with excellent results and limited late toxicity. Functioning scales were rated relatively highly in QoL questionnaires, and the overall global health score was comparable to published data. Gastrointestinal difficulties, fatigue, and sexual dysfunction dominated the symptom scales in our cohort.
- MeSH
- brachyterapie * metody MeSH
- celková dávka radioterapie MeSH
- dospělí MeSH
- kvalita života * psychologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádory anu * radioterapie psychologie patologie MeSH
- následné studie MeSH
- průzkumy a dotazníky MeSH
- radiační poranění * psychologie etiologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- spinocelulární karcinom * radioterapie patologie MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
β-Glucans comprise a group of β-D-glucose polysaccharides (glucans) that occur naturally in the cell walls of bacteria, fungi, and cereals. Its degradation is catalyzed by β-glucanases, enzymes that catalyze the breakdown of β-glucan into cello-oligosaccharides and glucose. These enzymes are classified as endo-glucanases, exo-glucanases, and glucosidases according to their mechanism of action, being the lichenases (β-1,3;1,4-glucanases, EC 3.2.1.73) one of them. Hence, we aimed to enhance lichenase production by Thermothelomyces thermophilus through the application of response surface methodology, using tamarind (Tamarindus indica) and jatoba (Hymenaea courbaril) seeds as carbon sources. The crude extract was immobilized, with a focus on improving lichenase activity, using various ionic supports, including MANAE (monoamine-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose, and PEI (polyethyleneimine)-agarose. Regarding lichenase, the optimal conditions yielding the highest activity were determined as 1.5% tamarind seeds, cultivation at 50 °C under static conditions for 72 h. Moreover, transitioning from Erlenmeyer flasks to a bioreactor proved pivotal, resulting in a 2.21-fold increase in activity. Biochemical characterization revealed an optimum temperature of 50 °C and pH of 6.5. However, sustained stability at varying pH and temperature levels was challenging, underscoring the necessity of immobilizing lichenase on ionic supports. Notably, CM-cellulose emerged as the most effective immobilization medium, exhibiting an activity of 1.01 U/g of the derivative (enzyme plus support), marking a substantial enhancement. This study marks the first lichenase immobilization on these chemical supports in existing literature.
- MeSH
- enzymy imobilizované * metabolismus chemie MeSH
- fungální proteiny * metabolismus chemie MeSH
- glykosidhydrolasy * metabolismus chemie biosyntéza MeSH
- koncentrace vodíkových iontů MeSH
- ovoce metabolismus MeSH
- semena rostlinná metabolismus MeSH
- Sordariales MeSH
- stabilita enzymů MeSH
- Tamarindus metabolismus mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: This study aimed to evaluate the beliefs about third-hand tobacco smoke (THS) among administrative and academic staff at a university. THS is a residual pollutant from tobacco smoke that lingers on surfaces and poses health risks, particularly to children. The study also aimed to understand these beliefs in relation to socio-demographic factors. METHODS: A cross-sectional survey was conducted with 332 university employees aged 18 and over. Data were collected between September and December 2023 through face-to-face and online questionnaires. The survey included the Beliefs About Third-hand Smoke (BATHS-T) Scale and questions on socio-demographic characteristics, second-hand smoke exposure, and attitudes towards a smoke-free campus. Beliefs About Third-hand Smoke Scale total and sub-dimension scores increase, it is understood that the participant's belief in the negative effects of third-hand tobacco exposure on persistence and health increases. RESULTS: The average age of participants was 36.1 years, with 38.3% being administrative personnel and 61.7% academic personnel. About 40.5% of the participants used tobacco products. The mean total BATHS-T score was 35.6, with health and persistence subscale scores averaging 19.9 and 15.7, respectively. Non-smokers had significantly higher BATHS-T scores than smokers. The participants with children scored higher on the health sub-dimension. Additionally, 78.9% supported a smoke-free campus, and those supporting it had higher BATHS-T scores. Awareness of the harms of second-hand smoke correlated with higher BATHS-T scores. CONCLUSION: The study highlights that non-smokers and those with children are more aware of THS risks. There is strong support for a smoke-free campus among university staff. The findings suggest a need for increased education on THS, especially targeted at smokers and those without children. Universities can play a crucial role in promoting smoke-free environments and raising awareness about the health risks associated with THS.
- MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- průřezové studie MeSH
- průzkumy a dotazníky MeSH
- univerzity MeSH
- zdraví - znalosti, postoje, praxe * MeSH
- znečištění tabákovým kouřem * škodlivé účinky statistika a číselné údaje MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.
- MeSH
- arabinosa metabolismus MeSH
- Bacillus subtilis * genetika enzymologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory genetika MeSH
- klonování DNA MeSH
- lipasa * genetika metabolismus MeSH
- Pseudomonas aeruginosa * genetika enzymologie MeSH
- rekombinantní proteiny * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production.
- MeSH
- asparaginasa * genetika biosyntéza metabolismus MeSH
- Escherichia coli K12 genetika enzymologie MeSH
- Escherichia coli genetika metabolismus MeSH
- fermentace MeSH
- isopropylthiogalaktosid farmakologie MeSH
- rekombinantní proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Tannase-producing filamentous fungi residing alongside tannin-rich ambient in the Northwest Himalayas were isolated at laboratory conditions and further identified by 18S ribosomal RNA gene sequencing. Five most potent tannase producing strains (EI ≥ 2.0), designated Aspergillus fumigatus AN1, Fusarium redolens AN2, Penicillium crustosum AN3, Penicillium restrictum AN4, and Penicillium commune AN5, were characterized. The strain Penicillium crustosum AN3 exhibited a maximum zone dia (25.66 mm ± 0.38). During solid-state fermentation, a maximal amount of tannase was attained with Penicillium crustosum AN3 using pine needles (substrate) by adopting response surface methodology for culture parameter optimization. Gel filtration chromatography yielded 46.48% of the partially purified enzyme with 3.94-fold of tannase purification. We found two subunits in enzyme-117.76 KDa and 88.51 KDa, respectively, in the SDS-PAGE. Furthermore, the characterization of partially purified tannase revealed a maximum enzyme activity of 8.36 U/mL at 30 °C using a substrate concentration (methyl gallate) of 10 mM. To broaden the knowledge of crude enzyme application, dye degradation studies were subjected to extracellular crude tannase from Penicillium crustosum AN3 where the maximum degradation achieved at a low enzyme concentration (5 ppm).
- MeSH
- barvicí látky metabolismus chemie MeSH
- fermentace MeSH
- fungální proteiny genetika metabolismus izolace a purifikace chemie MeSH
- Fusarium enzymologie genetika MeSH
- fylogeneze MeSH
- houby enzymologie genetika MeSH
- karboxylesterhydrolasy * metabolismus genetika izolace a purifikace chemie MeSH
- kultivační média chemie MeSH
- molekulová hmotnost MeSH
- Penicillium * enzymologie genetika MeSH
- RNA ribozomální 18S genetika MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND AND OBJECTIVE: The prevalence of pelvic floor muscle injuries induced by childbirth is higher than 23 % in the general women population. Such injuries can lead to prolapses and other pathologies in future female life. Leveraging computational biomechanics, the study implements an advanced female pelvic floor model for computing the maximum pelvic muscle strain, which serves as an injury risk indicator. The design of experiment method, abbreviated as DoE, is used to compute the maximum strain for boundary values of bony pelvis dimensions, namely the anterior-posterior diameter (abbreviated as APD) and the transverse diameter (abbreviated as TD). This is done in combination with small, medium and large percentiles of fetal head circumference (abbreviated as HC). METHODS: We utilized a previously developed finite element model of a female pelvic floor, as a reference, and enhanced it with new features, including a more detailed tissue geometry and advanced constitutive material models. The APD and TD dimensions were sourced from the set of MRI of 64 nulliparous women. This data was used to estimate the boundary dimensions of the female bony pelvis, combining both small and large values of APD and TD. Together with the 10th and the 95th percentiles for HC, a three-dimensional domain was constructed to assess the maximum pelvic muscle strain. In boundary cases, the maximum pelvic muscle strain was computed across 8 full-factorial design models (each situated at one corner of the domain, thereby combining the minimum and the maximum values of APD, TD and HC). This was done to define a response surface that predicts the maximum pelvic muscle strain within the domain. The accuracy of this response surface prediction was validated using 15 additional intermediate design models. These models were placed at the center of the domain (1 point), the centres of the domain boundary surfaces (6 points), and midway along each domain boundary edge (8 points). RESULTS: The maximum strain results for 8 combinations of APD, TD, and HC were employed to construct a linear response surface as a function of APD, TD, and HC. Tests at an additional 19 domain points served to evaluate the efficiency of the response surface prediction. The response surface demonstrated strong predictability, with an absolute average error of 1.52 %, an absolute median error of 1.52 %, and an absolute maximum error of 11.11 %. HC emerged as the most influencing dimension, accounting for 16 % of influence. CONCLUSIONS: The reference finite element pelvic floor model was scaled to 8 full-factorial female-specific pelvic floor models, which represent the combination of boundary values for APD, TD, and HC. The maximum pelvic floor muscle strain from these 8 models was used to design a response surface. When implementing the DoE approach to construct the response, there was consistent predictability for the maximum perineal muscle strain, as validated by the additional 19 intermediate design models. As a result, the response surface methodology can serve as an initial predictor for potential childbirth-induced pelvic floor muscle injury.
- MeSH
- kosterní svaly diagnostické zobrazování MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- pánevní dno diagnostické zobrazování fyziologie MeSH
- porod * fyziologie MeSH
- těhotenství MeSH
- vedení porodu * MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Nutrient deficiency induces a variety of cellular responses, including an increase in lipid accumulation in microalgae. Nitrogen starvation is the most studied deprivation. Here, we determine the effects of phosphorus and sulfur limitation on lipid accumulation in Chlorella vulgaris. A set of 9 experiments were performed, varying the initial concentration of these nutrients (set to 0, 50, and 100% of their original composition in Bold's basal medium). According to our results, the variation of P and S modified the specific growth rate, lag phase, and cell generation time. The ratio of 50%P and 0%S significantly increased the total lipid concentration. The fatty acid profile was dominated by C16:0, C18:0, and C18:1; a considerable increase in C20:5 was observed with 0%P and 50%S and 0%P and 100%S. Regarding neutral lipids, the response surface methodology (RSM) indicates that the maximum was observed when S was between 40 and 60% and P was between 95 and 100%. Therefore, the enhanced production of lipids caused by P and S limitation may contribute to the efficient oil production useful for algal biofuels.
Production of amylases by fungi under solid-state fermentation is considered the best methodology for commercial scaling that addresses the ever-escalating needs of the worldwide enzyme market. Here response surface methodology (RSM) was used for the optimization of process variables for α-amylase enzyme production from Trichoderma virens using watermelon rinds (WMR) under solid-state fermentation (SSF). The statistical model included four variables, each detected at two levels, followed by model development with partial purification and characterization of α-amylase. The partially purified α-amylase was characterized with regard to optimum pH, temperature, kinetic constant, and substrate specificity. The results indicated that both pH and moisture content had a significant effect (P < 0.05) on α-amylase production (880 U/g) under optimized process conditions at a 3-day incubation time, moisture content of 50%, 30 °C, and pH 6.98. Statistical optimization using RSM showed R2 values of 0.9934, demonstrating the validity of the model. Five α-amylases were separated by using DEAE-Sepharose and characterized with a wide range of optimized pH values (pH 4.5-9.0), temperature optima (40-60 °C), low Km values (2.27-3.3 mg/mL), and high substrate specificity toward large substrates. In conclusion, this study presents an efficient and green approach for utilization of agro-waste for production of the valuable α-amylase enzyme using RSM under SSF. RSM was particularly beneficial for the optimization and analysis of the effective process parameters.
